Use of Midostaurin for Treating Gastrointestinal Stromal Tumors

ABSTRACT

The present invention relates to the use of midostaurin, in free form or in pharmaceutically acceptable salt form in the manufacture of a pharmaceutical composition for the treatment of gastrointestinal stromal tumors, and to a method of treatment of warm-blooded animals, preferably humans, in which a therapeutically effective dose of midostaurin is administered to an animal suffering from said disease or condition.

The present invention relates to the use of midostaurin, in free form orin pharmaceutically acceptable salt form in the manufacture of apharmaceutical composition for the treatment of gastrointestinal stromaltumors, e.g. gastrointestinal tumors resistant to Compound I, and to amethod of treatment of warm-blooded animals, preferably humans, in whicha therapeutically effective dose of midostaurin animal suffering fromsaid disease or condition mentioned above.

Description of FIG. 1.

Panel B: dose response curves of imatinib or PKC412 for Ba/F3 cellsexpressing KIT ΔWK557-558/T670I, PDGFRA D842V or ΔDIM842-844 mutations.

Gastrointestinal stromal tumours are a recently characterized family ofmesenchymal neoplasms, which originate from the gastrointestinal tract,60 to 70% of all GISTs originate from the stomach. In the past, thesetumours were variously classified as leiomyoma, leiomyoblastoma, orleiomyosarcoma. However, it is now clear that GISTs represent a distinctclinicopathologic set of diseases based on their unique molecularpathogenesis and clinical features.

GIST is a relatively rare condition and has an estimated incidence ofabout 20 cases/million, GIST is the most common mesenchymal neoplasm ofthe gastrointestinal tract. Until recently the only available therapyhas been surgical resection. The limited value of conventional cytotoxicchemotherapy and radiation therapy has resulted in advanced GIST beingan invariably progressive and fatal condition, the median survival ofpatients varying from 20 months, e.g. metastatic GIST, to a year orless, e.g. post-surgical recurrence.

The most likely causative oncogenic molecular event in the vast majorityof GISTs is an activating mutation of KIT or platelet-derived growthfactor receptor A, abbreviated as PDGFRA. As a result signaling pathwaysare activated that promote cell proliferation and/or survival. Imatinibmesylate specifically inhibits the receptor tyrosine kinases PDGFRs,KIT, ABL, and ARG, and induces high response rates in patients withGISTs. To date, imatinib therapy remains the only effective, systemictreatment for this disease. Clinical and experimental observationslinked the response to the presence and the type of KIT/PDGFRA mutationsin the tumor, with those carrying KIT exon 11 mutations being the mostsensitive to treatment. KIT-D816V and PDGFRA-D842V mutations, affectingthe kinase catalytic domain, interfere with the binding of imatinib andrender the drug primary ineffective. The majority of GIST patientsdevelop resistance during therapy, after differing degrees of initialresponse to the drug. The investigation of other malignancies treatedwith imatinib, such as chronic myeloid leukemia (CML), or chroniceosinophilic leukemia (CEL), indicates that resistance to this inhibitorcan be caused by distinct molecular mechanisms. The majority of CMLpatients with imatinib-resistance have a clonal expansion of leukemiccells harboring novel mutant BCR-ABL alleles or expressing higher levelsof the fusion protein due to BCR-ABL amplification. The development ofresistance to imatinib in CEL can be associated with a secondarymutation within catalytic domain of FIPL1-PDGFRA fusion protein.Preliminary studies in GIST patients with imatinib-resistant progressivestage of disease indicated that in a majority of tumors KIT activationstill continued to play a functional role, with acquired mutations ofKIT kinase domain or genomic amplification of KIT gene as a causativefactors in a subset of patients.

Imatinib is a small molecule selectively inhibiting specific tyrosinekinases that has emerged recently as a valuable treatment for patientswith advanced GIST. The use of imatinib as monotherapy for the treatmentof GIST has been described in PCT publication WO 02/34727, which is hereincorporated by reference. However, it has been reported that primaryresistance to imatinib is present in a population of patients, forexample 13.7% of patients in one study. In addition, a number ofpatients acquire resistance to treatment with imatinib. More generallythis resistance is partial with progression in some lesions, butcontinuing disease control in other lesions. Hence, these patientsremain on imatinib treatment but with a clear need for additional oralternative therapy.

Imatinib is4-(4-methylpiperazin-1-ylmethyl)-N-[4-methyl-3-(4-pyridin-3-yl)pyrimidin-2-ylamino)phenyl]-benzamidehaving the formula I

The preparation of imatinib and the use thereof, especially as ananti-tumour agent, are described in Example 21 of European patentapplication EP-A-0 564 409, which was published on 6 Oct. 1993, and inequivalent applications and patents in numerous other countries, e.g. inU.S. Pat. No. 5,521,184 and in Japanese patent 2706682, all of which areincorporated by reference herein.

It has now surprisingly been found that midostaurin, a protein kinase Cinhibitor, possesses therapeutic properties which render it useful forthe treatment of gastrointestinal stromal tumors, e.g. for the treatmentof imatinib-resistant gastrointestinal stromal tumors.

Protein kinase C, herein after abbreviated as PKC, is one of the keyenzymes in cellular signal transduction pathways, and it has a pivotalrole in the control of cell proliferation and differentiation. PKC is afamily of serine/threonine kinases. At least 12 isoforms of PKC havebeen identified, and they are commonly divided into three groups basedon their structure and substrate requirements. PKC expression has beenfound to be elevated in human breast tumor biopsies as compared withnormal breast tissues, and high PKC expression has been considered as abiological marker for malignancy in human astrocytomas. One of the PKCisoforms, PKCθ, is a positive regulator of survival signaling in Tcells. Interestingly, PKCθ is constitutively phosphorylated in GIST.Thus, PKCθ may be considered a potential target kinase for therapeuticinterventions in GIST. In particular, PKC inhibitors are beneficial inthe treatment of imatinib resistant GISTs.

Accordingly, the present invention relates to a method of treating GIST,which comprises administering midostaurin, to a patient with GIST, e.g.with imatinib-resistant GIST.

Midostaurin according to the invention isN-[(9S,10R,11R,13R)-2,3,10,11,12,13-hexahydro-10-methoxy-9-methyl-1-oxo-9,13-epoxy-1H,9H-diindolo[1,2,3-gh:3′,2′,1′-lm]pyrrolo[3,4-j][1,7]benzodiazonin-11-yl]-N-methylbenzamideof the formula (II):

or a salt thereof, hereinafter: “Compound of formula II or midostaurin”.

Compound of formula II or midostaurin [International NonproprietaryName] is also known as PKC412.

Midostaurin is a derivative of the naturally occurring alkaloidstaurosporine, and has been specifically described in the Europeanpatent No. 0 296 110 published on Dec. 21, 1988, as well as in U.S. Pat.No. 5,093,330 published on Mar. 3, 1992, and Japanese Patent No. 2 708047. Midostaurin described in these documents are incorporated into thepresent application by reference. Midostaurin and its manufacturingprocess has been specifically described in many documents, well known bythe man skilled in the art.

In each case where citations of patent applications or scientificpublications are given in particular for midostaurin, the subject-matterof the final products, the pharmaceutical preparations and the claimsare hereby incorporated into the present application by reference tothese publications.

The term “imatinib-resistant or imatinib-resistance” as used hereindefines a lack, a reduction or a loss of therapeutic effectiveness ofimatinib in the treatment of gastrointestinal stromal tumors.

The invention relates to the use of midostaurin, also known as PKC412,or a pharmaceutically acceptable salt thereof, for the manufacture of amedicament for the treatment of gastrointestinal stromal tumours, hereinafter abbreviated as GIST, e.g. imatinib-resistant GIST, and to a methodof treating warm-blooded animals, including humans, suffering from GISTby administering to a said animal in need of such treatment an effectiveamount of midostaurin, or a pharmaceutically acceptable salt thereof.

The present invention relates to a method of treating GIST, e.g. withimatinib-resistant GIST, which comprises administering midostaurin, to apatient with GIST, e.g. with imatinib-resistant GIST.

The precise dosage of midostaurin to be employed for treating thediseases and conditions mentioned hereinbefore depends upon severalfactors including the host, the nature and the severity of the conditionbeing treated, the mode of administration. In general, satisfactoryresults are achieved when midostaurin is administered parenterally,e.g., intraperitoneally, intravenously, intramuscularly, subcutaneously,intratumorally, or rectally, or enterally, e.g., orally, preferablyintravenously or, preferably orally, intravenously at a daily dosage of0.1 to 10 mg/kg body weight, preferably 1 to 5 mg/kg body weight. Inhuman trials a total dose of 225 mg/day was most presumably the MaximumTolerated Dose (MTD). A preferred intravenous daily dosage is 0.1 to 10mg/kg body weight or, for most larger primates, a daily dosage of200-300 mg. A typical intravenous dosage is 3 to 5 mg/kg, three to fivetimes a week.

Midostaurin is administered orally in dosages up to about 300 mg/day,for example 100 to 300 mg/day. The midostaurin is administered as asingle dose or split into two or three doses daily, preferably twodoses. A particularly important dose is 200-225 mg/day, in particular100 mg twice a day (200 mg/day total). The upper limit of dosage is thatimposed by side effects and can be determined by trial for the patientbeing treated.

The instant invention also concerns a method wherein the therapeuticallyeffective amount of midostaurin is administered to a mammal subject 7 to4 times a week or about 100% to about 50% of the days in the timeperiod, for a period of from one to six weeks, followed by a period ofone to three weeks, wherein the agent is not administered and this cyclebeing repeated for from 1 to several cycles.

Usually, a small dose is administered initially and the dosage isgradually increased until the optimal dosage for the host undertreatment is determined. The upper limit of dosage is that imposed byside effects and can be determined by trial for the host being treated.

Midostaurin may be combined with one or more pharmaceutically acceptablecarriers and, optionally, one or more other conventional pharmaceuticaladjuvants and administered enterally, e.g. orally, in the form oftablets, capsules, caplets, etc. or parenterally, e.g.,intraperitoneally or intravenously, in the form of sterile injectablesolutions or suspensions. The enteral and parenteral compositions may beprepared by conventional means.

The infusion solutions according to the present invention are preferablysterile. This may be readily accomplished, e.g. by filtration throughsterile filtration membranes. Aseptic formation of any composition inliquid form, the aseptic filling of vials and/or combining apharmaceutical composition of the present invention with a suitablediluent under aseptic conditions are well known to the skilledaddressee.

Midostaurin may be formulated into enteral and parenteral pharmaceuticalcompositions containing an amount of the active substance that iseffective for treating the diseases and conditions named hereinbefore,such compositions in unit dosage form and such compositions comprising apharmaceutically acceptable carrier.

Examples of useful compositions are described in the European patentsNo. 0 296 110, No. 0 657 164, No. 0 296 110, No. 0 733 372, No. 0 711556, No. 0 711 557.

The preferred compositions are described in the European patent No. 0657 164 published on Jun. 14, 1995. The described pharmaceuticalcompositions comprise a solution or dispersion of midostaurin in asaturated polyalkylene glycol glyceride, in which the glycol glycerideis a mixture of glyceryl and polyethylene glycol esters of one or moreC₈-C₁₈ saturated fatty acids.

The present invention relates to the use of midostaurin, or apharmaceutically acceptable salt thereof for the preparation of amedicament for the treatment of GIST, e.g. imatinib-resistant GIST, withthe proviso that midostaurin is not administered together, sequentially,or separately with imatinib.

The present invention relates to the use of midostaurin or apharmaceutically acceptable salt thereof for the treatment of GIST, e.g.imatinib-resistant GIST, wherein imatinib is not used for the treatmentof said GIST, e.g. imatinib-resistant GIST.

The present invention relates to the use of midostaurin or apharmaceutically acceptable salt thereof wherein midostaurin is used asan anti-tumor agent for the treatment of GIST, e.g. imatinib-resistantGIST.

The present invention further relates to packaged midostaurin whatincludes instructions to use midostaurin, or salts thereof, together forthe treatment of GIST, e.g. imatinib-resistant GIST.

In one aspect the present invention provides a method of treating GISTcomprising administering midostaurin in an amount which istherapeutically effective against GIST to a warm-blooded animal,particularly a human, in need thereof. More particularly, the presentinvention provides a method of treating a patient suffering from GIST,which comprises administering an effective amount of midostaurin, or apharmaceutically acceptable salt thereof, to the patient. Moreparticularly, the present invention provides a method of treating apatient suffering from GIST, which comprises administering an effectivemidostaurin, or a pharmaceutically acceptable salt thereof, to thepatient, wherein the midostaurin is administered in a dose of 100 to 300mg daily, particularly 150 to 250 mg daily, most particularly 200 mgdaily, as an oral pharmaceutical preparation

EXAMPLE 1 Midostaurin Pharmaceutical Preparations Composition A:

Gelucire 44/14 (82 parts) is melted by heating to 60° C. PowderedMidostaurin (18 parts) is added to the molten material. The resultingmixture is homogenised and the dispersion obtained is introduced intohard gelatin capsules of different size, so that some contain a 25 mgdosage and others a 75 mg dosage of the Midostaurin. The resultingcapsules are suitable for oral administration.

Composition B:

Gelucire 44/14 (86 parts) is melted by heating to 60° C. PowderedMidostaurin (14 parts) is added to the molten material. The mixture ishomogenised and the dispersion obtained is introduced into hard gelatincapsules of different size, so that some contain a 25 mg dosage andothers a 75 mg dosage of the Midostaurin. The resulting capsules aresuitable for oral administration.

Gelucire 44/14 available commercially from Gattefossé; is a mixture ofesters of C8-C18 saturated fatty acids with glycerol and a polyethyleneglycol having a molecular weight of about 1500, the specifications forthe composition of the fatty acid component being, by weight, 4-10%caprylic acid, 3-9% capric acid, 40-50% lauric acid, 14-24% myristicacid, 4-14% palmitic acid and 5-15% stearic acid.

A preferred example of Gelucire formulation consists of:

Gelucire (44/14): 47 g

Midostaurin: 3.0 g filled into a 60 mL Twist off flask

Composition C: an Example of Soft Gel Will Contain the FollowingMicroemulsion:

Cornoil glycerides 85.0 mg Polyethylenglykol 400 128.25 mg Cremophor RH40 213.75 mg Midostaurin 25.0 mg DL alpha Tocopherol 0.5 mg Ethanolabsolute 33.9 mg Total 486.4 mg

EXAMPLE 2

PKC412 interacts strongly with ATP binding sites of the conventionalPKCs, FLT3, PDGFRs, VEGFRs, KIT and the CDK1-cyclin B complex. Notably,PKC412 was shown to exhibit full inhibitory activity against theimatinib-resistant T674 I mutant form of FIPL1-PDGFRA in refractory CELpatients, see e.g. Cools J., et al., Cancer Cell 2003; 3:459-469. Thecatalytic sites of tyrosine kinases are highly conserved, and the T674Imutation in PDGFRA corresponds to the T315I mutation in ABL and theT670I mutation in KIT, the resistant mutations in progressive BCR-ABLpositive CML and in KIT mutant GISTs patients, respectively. Themechanisms of resistance to imatinib in 26 patients with GISTsrefractory to imatinib is investigated and the use of PKC412 to overcomethe clinical resistance to imatinib in those patients due to therecurrent KIT-T670I or -V654A, and PDGFRA-D842V kinase domain mutationsis explored.

Materials and Methods

Patients: Progressive tumors from 26 patients treated with imatinib inthe Department of Oncology, University Hospital Leuven were evaluated.There are 20 men and 6 women, with a median age of 53 years (range, 37to 77 years). Twenty-two out of 26 patients had the primary tumorsurgically removed. Chemotherapy and/or radiotherapy was applied in theadvanced stage of the disease in 13 patients, prior to imatinibtreatment. Patients whose tumor progressed but who were otherwise ingood clinical condition were eligible to dose increase Up to 1000 mgdaily. Dose escalation decisions were based on data from patientstreated at least 4 weeks. Lesions were reassessed after one month, threemonths, and every six months thereafter. Progression was based onclinical examination and CT/PET imaging, and defined according tocriteria previously published, see e.g. Van Oosterom AT et al., Lancet2001; 358:1421-1423. Histopatlhological and molecular changes during thetreatment are evaluated in selected consenting patients by means ofserial tumor biopsies.

Pathology: Histopathologic and immunohistochemical analyses areperformed on tissue embedded in paraffin. Polyclonal antibodies againstCD117 (A4502, dilution 1/250, DAKO, Denmark) andavidin-biotin-peroxidase complexes are used without any antigenretrieval.

Fluorescence in situ hybidization (FISH). Dual-color interphase FISHanalysis is performed on 4 μm paraffin embedded tissue sections of tumorbiopsies obtained before imatinib treatment (18 cases), or on touchpreparations from fresh biopsies of imatinib-resistant lesions (all 26cases). Digoxigenin- or biotin labeled BAC clones for KIT/4q12(RP11-568A2) or PDGFRA/4q12 (RP11-24O11) are co-hybridized withSpectrumGreen- or SpectrumOrange-labeled chromosome 4 centromeric probes(CEP4, Vysis Inc., Downers Grove, Ill., USA), respectively, aspreviously described. 21 The FISH data are collected on a Leica DMRB(Leica, Wetzlar, Germany) fluorescence microscope equipped with a cooledblack and white charged couple device camera (Photometrics, Tuscon,Ariz.), run by Quips SmartCapture™ FISH Imaging Software (Vysis,Bergisch-Gladbach, Germany). eHundred interphase nuclei are evaluated,and the ratio of KIT/PDGFRA to CEP4 was calculated. A ratio of ≧2 isdefined as specific KIT/PDGFRA amplification.

Sequence analysis: Genomic DNA is extracted from snap-frozen tissueusing the High Pure PCR Template Preparation Kit (Roche, Mannheim,Germany) Exons 9, 11, 13, 14, 15 and 17 of the KIT, and exons 12 and 18of the PDGFRA are amplified by the polymerase chain reaction (PCR) aspreviously described, see e.g. Debiec-Rychter M et al., J Pathol 2004;202:430-438. The PCR products were purified (Microcon PCR, Millipore,Mass., USA) and screened for mutations by denaturing high-performanceliquid chromatography on a Transgenomic WAVE DHPLC system (DIIPLC;Transgenomic, Inc., UK). Samples showing an aberrant elution profilewere re-amplified and sequenced.

Western-blot: Snap-frozen tumor specimens sufficient for preparation ofcell lysates were available from ten refractory GISTs. Cell lysis,SDS-PAGE and immunoblotting were carried out as described.²¹ Membranes(Amnersham Pharmacia Biotechnology, UK) were immunoblotted overnightusing anti-phospho-KIT (Y703) (Zymed, San Francisco, Calif.) antibody atdilution of 1:500. The HRP-conjugated anti-rabbit IgG was used at adilution of 1:2500 and visualized with Enhanced Chemiluminescence(Pierce). Membranes were then stripped and re-blotted to determine totalprotein levels using an antibody recognizing total KIT protein(anti-CD117, A4502, DAKO, Glostrup, Denmark).

Primary resistant GIST cells response assay: Imatinib mesylate andPKC412, the crystalline compounds are dissolved at 10 mM in 100% DMSO(Sigma) and aliquots are kept at −80° C. Experiments are performed withserial dilutions of the 10 mM stock. Controls are performed with solvent(DMSO) dilutions. Primary cells are obtained from collagenasedisaggregated progressive tumor specimens, seeded at 60-70% confluencein 100-mm cell culture dishes (Corning Inc., Corning, N.Y.) and grownfor three days in DMEM supplemented with 10% fetal bovine serum, 0.1 mMnonessential amino acids, and 1.0 mM sodium pyruvate. Cells are exposedto either imatinib mesylate, PKC412 or vehicle alone for 90 min, washedwith 10 ml of cold PBS, and lysed in buffer [1% NP40, 50 mM Tris-HCl pH8.0, 150 mM NaCl, supplemented with complete protease inhibitor cocktailtablets (Boehringer Mannheim GmbH, Mannheim, Germany) and 0.2 mM sodiumorthovanadate (Sigma, St. Louis, Mo.)].

Construct: Mutant PDGFRA and KIT cDNA are obtained by RT-PCR on RNAisolated from progressive tumors. The cDNA's are cloned into theretroviral vector pMSCV-puro (Clontech).

Cell culture: 293T cells are grown in DMEM supplemented with 10% FCS.Ba/F3 cells are grown in RPMI-1640 supplemented with 10% FCS andinterleukin-3 (1 ng/ml). Virus as produced as described previously, seee.g. Cools J. et al., N Engl J Med 2003; 348:1201-1214.

.Ba/F3 cells transduced with the different constructs are selected withpuromycin (2 μg/ml). To test for factor independent growth, Ba/F3 cellsare washed 3 times in PBS and new cultures are initiated in the absenceof interleukin-3. Cells that became independent on interleukin-3, aremaintained in the absence of interleukin-3. For dose-response curves,Ba/F3 cells are grown in 24-well plates with different concentrations ofinhibitor. The number of viable cells is determined at the start andafter 24 hrs, using the AqueousOne solution (Promega).

Results: Progressive tumors from 26 patients treated with imatinib areevaluated. The median time from the diagnosis to the proven malignancyof the disease is 48 weeks (range, 0 to 265 weeks), while the mediantime from the diagnosis to imatinib treatment is 91 weeks (range, 6 to304 weeks). Fifteen patients (57.6%) achieved partial remission, and 10patients (38.4%) showed stable disease during imatinib treatment, withan average duration of event free survival of 48 weeks (range 16 to 200weeks).

Histopathology: Twenty-five primary GISTs reveal spindle cell and onehad mixed morphology. CD117 antigen expression is demonstrated in eachprimary tumor and in 24 out of 26 (92%) progressive biopsies. Twoimatinib-resistant GISTs invert their histologic appearance from spindleto epithelioid type and their immunophenotype, becoming CD117 negative(data not shown).

Mutation analysis: A combination of D-HPLC and direct sequencingrevealed KIT mutations in 25 out of 26 (96.1%) base-line GIST biopsies,see Table 1. Nineteen tumors harbored exon 11 juxtamembrane mutationsand six carried exon 9 mutations. None pre-treatment tumor specimen hadmutations in PDGFRA or more than one mutation in KIT. One tumor had noidentifiable KIT or PDGFRA sequence alteration in the examined exons.While no point mutations of the KIT kinase domain are detected in thetumors before imatinib treatment, six distinct secondary KIT mutationsare identified in 12/26 (48%) patients at the time of progression, aftera median of 77 weeks (range 16-188) on therapy. Four patients had aV654A and three patients had a T670I substitution, while the remainingpatients carried D716N, D816G, D820Y, D820E or N822K mutations. Onepatient with an original KIT G565R mutation acquired a D842V pointmutation in PDGFRA, not detectable in the primary tumor from thispatient.

FISH analysis. FISH analyses reveal amplification of KIT in 2 of 26(7.7%) progressive tumors. In the primary non-responding tumor frompatient 26, KIT amplification is associated with simultaneousamplification of PDGFRA (data not shown). No KIT or PDGFRA mutations arefound in the tumor from this patient, neither before treatment norduring progression of the disease. In one patient, KIT amplification (upto 5-fold) is not associated with increased PDGFRA copy number. Thiscase harbored a primary KIT mutation, but secondary mutations are notidentified during progression. In six imatinib-resistant specimens, lossof KIT/PDGFRA/CEP4 loci is revealed by interphase FISH analysis. Whilein three of the tumors, this hemizygosity is already observed in thebase-line tumor biopsies, in three other specimens, it is only presentin the progressive lesions. Within the latter, however, markedheterogeneity in the number of KIT/CEP4 signals per nuclei isencountered (range from 0 to 4). Particularly, 23% of cells inprogressive tumor biopsies from one patient showed bi-allelic loss ofKIT/PDGFRA/CEP4.

KIT activation in resistant GISTs. KIT activation in 10imatinib-resistant GISTs is evaluated by Western blotting withantibodies to KIT phosphotyrosine Y703 and total KIT. Eight specimensdemonstrate KIT expression and various levels of constitutive KITautophosphorylation. Four of these eight tumors have secondary KITmutations, and for the remaining four the reason for the re-activationof KIT in imatinib-resistant tumor cells is unknown. Two resistantmetastatic tumors totally lacked KIT expression, which is in line withthe loss of CD117-positivity by immunohistochemistry, and the observedbi-alleic loss of KIT loci in one case.

Ex-vivo espouse of resistant GISTs to imatinib and PKC412: The effect ofimatinib and PKC412 on the autophosphorylation of the KIT Y703 residuein cultured imatinib-resistant cells that harbored KITΔ557-558/T670I orKITInsAY502-503/V654A mutant isoforms is determined by Western blot. Theresults are compared with GIST882 cells, which carry a hemizygous KITK642E mutation. Observations are standardized for total KIT expressionusing anti-KIT antibody. KIT protein is expressed and phosphorylated toa significant level in both resistant KITΔ557-558/T670I andKITIns503AY/V654A tumors and their in vitro cultured cell counterparts.The autophosphorylation of KIT is not affected by exposure of eitherprimary cell line to imatinib (up to 5 μM). In contrast, 0.5 μM PKC412reduced and 1 μM PKC412 totally inhibit KIT autophosphorylation of themutant KITΔ557-558/T670I cells. Similarly, KIT autophosphorylation ofthe mutant KITIns503AY/V654A is reduced by PKC412 already atconcentration 0.5 μM and completely inhibited at a ten-fold higherconcentration of the drug.

Effect of imatinib and PKC412 on KIT and PDGFRA mutants in vitro: Mutantforms of KIT Δ557-558/T670I, and PDGFRA ΔDIM842-844 and D842V areexpressed in Ba/F3 murine cells. Ba/F3 cells are IL3 dependent for theirgrowth, but become IL3 independent upon the expression of many activatedkinases, such as FIP1 L1-PDGFRA and BCR-ABL. Mutant KIT and PDGFRAproteins introduced in the Ba/F3 cells also confer factor independentgrowth, and are constitutively phosphorylated, confirming that these areactivated kinases (data not shown). Dose response curves and analysis ofthe phosphorylation state of KITΔ557-558/T670I with imatinib confirmedthe resistance to imatinib, with phosphorylation not completelyinhibited at 10 μM imatinib (cellular IC₅₀˜5 μM). The PDGFRA D824Vmutant also show resistance to imatinib, although to a lesser extent(cellular IC₅₀˜1 μM). The PDGFRA ΔDIM842-844 mutant serve as a controlin this experiment. All 3 mutants are inhibited by PKC412 atconcentrations below 1 μM, with PDGFRA D842V having the highest cellularIC₅₀ value of ˜200 nM (FIG. 1).

Preliminary studies described two categories of imatinib resistance:KIT-dependent or KIT-independent Mechanisms. ¹⁵ Based on our results, weconclude that re-activation of KIT is the most important mechanism forresistance. KIT is found to be phosphorylated (activated) in 8 of 10progressive tumors that could be analyzed by Western blot duringimatinib treatment. In 50% of these cases, reactivation of KIT is theconsequence of secondary resistance mutations, while in the other 50%the cause for reactivation remains unknown. Sequencing KIT in itsentirety in these samples may identify novel mutations in unexpectedregions of KIT that render the protein insensitive to imatinibtreatment. Alternatively, factors influencing intracellular drugdelivery or clearance could result in inadequate receptor inhibition,with a consequent progression of the disease.

In the 26 patients in our study, acquired secondary KIT mutations arethe most frequent event (48% of the cases) explaining resistance toimatinib. Six distinct secondary KIT mutations are identified inprogressive tumors. All are single amino acid substitutions and all arepresent in addition to the activating KIT mutations identified in thebase-line, non-treated tumors. To our knowledge, two recurrent KITmutations, V654A and T670I, and three others, D716N, D820E and D816G,present in single cases, have not been previously reported in primaryGISTs. This supports the close association of these mutations with thedevelopment of resistance to the drug. The D820Y and N822K mutations arepreviously described in imatinib non-treated GISTs. The activation loopmutations, e.g. D816G, D820E/Y, N822K, are likely to be activatingmutations in KIT that also directly confer resistance to imatinib. TheKIT D816V mutation in patients with systemic mastocytosis and in asubset of seminomas is associated with primary resistance to imatinib.

One tumor with a primary KIT G565R mutation acquires resistance toimatinib through a secondary PDGFRA D842V mutation. The D842V mutationis the most common activating PDGFRA mutation in GISTs, and is alsoproven to be imatinib-resistant. This mutation is an activating mutationthat shows decreased sensitivity to imatinib. The observation thatresistance to imatinib can occur through mutation of a different kinase,e.g. PDGFRA, identifies a previously not described mechanism ofresistance. In general, resistance of a tumor dependent on an activatedkinase sensitive to a small molecule inhibitor could occur by anactivating mutation in a different kinase that is not sensitive to thisinhibitor. It remains to be determined if this mechanism of resistanceoperates more frequently in GISTs and other tumors and leukemias, andwhether it is the cause of resistance in the cases of our study in whichwe are unable to identify secondary genomic changes in KIT.

In two cases of this study, imatinib-resistance is associated withamplification of KIT or KIT/PDGFRA genes. In the latter, the patientshowed primary resistance to imatinib with the massive progressive tumorgrowth, and consequently died five weeks from the start of imatinibadministration. As the malignant stage of the disease in this patientlasted over one year and the patient was pretreated with high dosechemo- and radiotherapy before treatment with imatinib, theamplification was most likely already present in tumor cells beforeimatinib administration and further selected for in the presence of thedrug. This finding indicates that KIT amplification may cause primaryresistance, and cautions the use of classical chemotherapy in GISTspatients, which may add to the evolution of the clonal diversityassociated with disease progression, with possible generation of thegenetic changes influencing the response to the drug.

Two progressive tumors completely lost KIT expression, indicatingKIT-independent mechanism of resistance. Interphase FISH analysisrevealed selective growth of cells with the bi-allelic loss of targetedKIT/PDGFRA genes in one of these tumors, further underlining the escapefrom the receptor dependence. The shift to KIT/PDGFRA hemizygosity isobserved in two tumors at the time of resistance to imatinib, which isassociated with the appearance of secondary KIT mutations. Whetherhemizygosity/homozygosity adds to insensitivity of recurrent mutants toimatinib is unclear and warrants further study.

In an attempt to define the imatinib sensitivity of the common KIT V654Aand T670I mutations present in tumor cells at the time of progression,the inhibitory effect of imatinib on the ligand-independent KITphosphorylation in cells harboring these mutations is examed using exvivo assay. In both cases, KIT autophosphorylation is not inhibited atconcentrations of imatinib as high as 5 μM, which is about the maximumlevel of imatinib that can be achieved in vivo. PKC412, an alternativeKIT and PDGFR inhibitor, exerted inhibitory effect on both mutants atthe concentrations that justify therapeutical use of the drug. Thedifferential sensitivity to imatinib and PKC412 on KIT T670I mutant isfurther validated in vitro using transformed Ba/F3 murine cells. Tofurther explore the sensitivity of other imatinib-resistant mutations toPKC412, Ba/F3 cells transfected with imatinib-resistant PDGFRA D842Vmutant are tested. PKC412 efficiently inhibits the PDGFRA D842V mutantat the concentration of 1 μM, additionally emphasizing the in vitropotency of the drug for inhibition of tumors harboring differentimatinib-resistant mutant isoforms. The existence of KIT-dependent andindependent mechanisms of imatinib-resistance in GISTs patients isconfirmed and reveals novel imatinib-resistant KIT mutant isoforms. Itpoints to the acquisition of imatinib-resistant PDGFRA mutations as acause of secondary resistance in a KIT positive tumor, and indicates theKIT amplification as the possible explanation not only for a secondarybut also for a primary resistance to the drug. The sensitivity of KITT670I and V654A, and PDGFRA D6842V mutations to PKC421 is evidenced.Given that individual kinase domain mutations exhibit differentialsensitivity to alternative kinase inhibitors, it is crucial to tailorsecond-line therapy precisely to the underlying mechanism of resistance.

TABLE 1 KIT and PDGFRA tumor genotype 26 GISTs patients. GenotypeBase-line biopsy Secondary mutations ^(a) Case KIT KIT or PDGFRA 1 PMK558N 2 Del WK557-558 3 Del WK557-558 ^(d) 4 Del WK557-558 ^(d) 5 DelKVVE558-561 6 Del KVVEEI 558-563 7 Del VYIDPTQL 569-576 8 DelGNNYVYIDPTQLPYD565-579V 9 PM V559G KIT V654A (GTG→GCG) 10 PM L576P ^(d)KIT V654A (GTG→GCG) ^(d) 11 Ins 574PT KIT V654A (GTG→GCG) 12 DelWK557-558 KIT D716N (GAT→AAT) 13 Del WK557-558 KIT T670I (ACA→ATA) 14Del WK557-558 KIT T670I (ACA→ATA) ^(d) 15 Del KPMYEVQWK 550-558Q KITT670I (ACA→ATA) 16 Del VEEINGNNYVYIDPTQL560-576 KIT D820E (GAT→GAA) 17Del VYIDPTQL 569-576 KIT D820Y (GAT→TAT) 18 Del VYIDPTQL 569-576 KITN822K (AAT→AAA) ^(d) 19 Ins 503AY KIT V654A (GTG→GCG) 20 Ins 503AY KITD816G (GAC→GGC) 21 Ins 503AY 22 Ins 503AY 23 Ins 503AY 24 Ins 503AY 25PM G565R PDGFRA D842V (GAC→GTC) 26 WT Abbreviations: WT—wild type; ^(a)mutations detected on the top of base-line mutant isoform; ^(b) range ofKIT signals per nucleus; ^(d) hemizygous by sequencing

1. A method of treating a patient suffering from gastrointestinalstromal tumors, which comprises administering an effective amount ofmidostaurin of formula,

or a pharmaceutically acceptable salt thereof, to the patient in needthereof.
 2. The method of claim 1 wherein the gastrointestinal stromaltumor is imatinib-resistant gastrointestinal stromal tumor.
 3. Themethod of claim 2 wherein the midostaurin is administered in a dose of100 to 300 mg daily.
 4. The method of claim 3 wherein the dose is 150 to250 mg daily.
 5. The method of claim 4 wherein the dose is 200 mg daily.6. The method of claim 1 wherein midostaurin is administered to thepatient with the proviso that midostaurin is not to be used forsimultaneous, separate or sequential use with imatinib.
 7. Use ofmidostaurin for the preparation of a medicament for the treatment ofgastrointestinal stromal tumors.
 8. The use according to claim 7 whereinthe gastrointestinal stromal tumors are resistant to therapy withimatinib.
 9. The use of claim 8 wherein the midostaurin is to beadministered in a dose from 150 to 250 mg daily.
 10. The use of claim 9wherein the dose to be administered is 200 mg daily.
 11. The method ofclaim 1 wherein the midostaurin is administered orally.
 12. The use ofclaim 7 wherein the midostaurin is administered orally.
 13. The methodof claim 2 wherein midostaurin is administered to the patient with theproviso that midostaurin is not to be used for simultaneous, separate orsequential use with imatinib.
 14. The method of claim 3 whereinmidostaurin is administered to the patient with the proviso thatmidostaurin is not to be used for simultaneous, separate or sequentialuse with imatinib.